The long term goals of this proposal are to establish the kinetic, thermodynamic and structural basis for force production in microtubule- dependent motility. Kinesin, the putative anterograde motor in axoplasmic transport, will be examined because it is the most simple of the ATPases involved in force production for sliding filament motility. Kinesin will be purified from cloned and overexpressed gene of Drosophila kinesin heavy chains or as native protein, form bovine brain. Conditions will be optimized for expression and purification of the kinesin head fragments, consisting of amino acids 10447 of Drosophila kinesin heavy chain. The microtubule-kinesin complex will be characterized to determine the stoichiometry of kinesin binding to tubulin and to establish conditions for kinetic and equilibrium measurements on the formation of the complex. The kinetics and thermodynamics of the microtubule-kinesin ATPase pathway will be established by transient state kinetic methods. Stopped-flow light scattering methods will be used to measure the rate of ATP-induced dissociation and reformation of the microtubule-kinesin complex. Chemical- quench-flow methods will be used to determine the rates of ATP binding and hydrolysis and ATP synthesis. These kinetic measurements will be extended to examine the potential regulation of kinesin by Ca+2 or by phosphorylation. Site-directed mutagenesis will be used to relate structural domains of the kinesin head to enzymatic functions responsible for energy transduction. Mutation of residues thought to be involved in ATP- or microtubule-binding or in regulation will allow a direct quantitative assessment of the roles of individual or groups of amino acids in each step in the ATPase cycle. Attempts will be made to grow crystals of the cloned kinesin head fragment, in order solve the structure of the kinesin head in the presence and absence of ATP and thereby define in molecular detail the nature of the conformational changes responsible for movement. Microtubule-dependent ATPases have been implicated in a large number of fundamental life processes including anterograde and retrograde axoplasmic transport in neurons and in chromosome movement during cell division. Thus, these studies into the fundamental molecular events leading to generation and control of force production have a wide applicability to health related research.